Embryonic stem (ES) cells can purportedly grow indefinitely while maintaining pluripotency and can differentiate into cells of all three germ layers, i.e., mesoderm, endoderm and ectoderm (Evans & Kaufman, Nature 292: 154-156 (1981)). Human ES cells and cells derived therefrom are currently being assessed for the treatment of a host of diseases, such as Parkinson's disease, spinal cord injury and diabetes. However, the fact that human ES cells are obtained from human embryos raises a number of highly contentious ethical considerations and in many countries the derivation of these cells is prohibited by law. Furthermore, because ES cells and cells derived therefrom express antigens from the subject from which they are derived, there is a risk that those cells will be rejected if administered to an unmatched (e.g., not expressing similar HLA type(s) subject. Accordingly, scientists have sought technical solutions to avoid the current methods of generating ES cells. One desirable way to accomplish these solutions would be to generate pluripotent cells directly from somatic cells of a post-natal individual, e.g., directly from a subject to be treated or a related or otherwise matched subject.
One method for reprogramming a somatic cells involves transferring the nuclear contents of the cell into an oocyte (Wilmut et al, Nature 385:810-813 (1997)) or by fusion with an ES cell (Cowan et al, Science 309: 1369-1373 (2005)), indicating that unfertilized eggs and ES cells contain factors that confer totipotency or pluripotency in somatic cells. Difficulties associated with these methods include the requirement for destruction of ova and/or embryos, which may raise ethical considerations in some countries.
Although the transcriptional determination of pluripotency is not fully understood, several transcription factors, including Oct 3/4 (Nichols et al, Cell 95:379-391 (1998)), Sox2 (Avilion et al, Genes Dev. 17: 126-140 (2003)) and Nanog (Chambers et al, Cell 113:643-655 (2003)) are involved in maintaining ES cell pluripotency; however, none is sufficient alone to specify ES cell identity.
Recently, Takahashi & Yamanaka introduced four factors (i.e., Oct4, Sox2, c-Myc and Klf4) into mouse ES cells and mouse adult fibroblasts cultured under conditions suitable for mouse ES cell culture. Following transduction into either cell type, the authors obtained induced pluripotent stem (iPS) cells that exhibited mouse ES cell morphology and growth properties and expressed mouse ES cell marker genes (Takahashi & Yamanaka, Cell 126:663-676 (2006)). Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. These data demonstrate that pluripotent cells can be directly generated from mouse fibroblast cultures by adding only a few defined factors using a retroviral transduction. However, this technique does suffer from some major disadvantages, including a low rate of reprogramming (considerably less than 1% of treated cells), and the need for genomic integration and continuous expression of the oncogenes c-Myc and Klf4. Expression of these genes may lead to production of tumors in recipients of the cells or cells derived therefrom. In this respect, chimeric mice produced using iPS cells generated with these methods develop tumors, presumably as a result of continuous expression of these oncogenes. Consequently, a major goal of research in the field is to develop reprogramming methods that either do not require genomic integration of nucleic acids encoding these factors or that minimize the number or duration of expression of these and other reprogramming factors.
While the majority of iPS cell-based studies use fibroblast cells, most likely due to their ease of derivation and extensive use in fusion-based reprogramming studies various cell populations have been used for iPS cell induction in the mouse other than fibroblasts. An important observation from these studies is that the somatic cell type selected had a significant effect on the efficiency of iPS cell generation and level of reprogramming. In this regard, some cell types, such as neural stem cells, stomach cells and liver cells appear to reprogram at relatively high efficiency compared to fibroblast cells. However, isolation of these cells from humans is difficult or not feasible due to the invasive nature of tissue collection and/or limited donor samples available. Some more accessible cell types (e.g., muscle cells or differentiated hematopoietic cells) have been used as the basis for iPS studies, however reprogramming has met with limited success.
Accordingly, there is a need in the art for identifying optimal cell types that are easily accessible and that reliably enable efficient reprogramming. Identifying such a cell type with efficient reprogramming properties would facilitate use of reprogramming methods without the need for genomic integration and/or with a minimum or reduced number of reprogramming factors. This would result in a safer pluripotent iPS cell population with reduced risk for neoplastic transformation. Such cell types, highly efficient for reprogramming and obtained without relying upon embryonic tissues, would be suited for use in applications already contemplated for existing, pluripotent ES cells.